Nucleic acid structure

1. STRUCTURE OF DNA Dr. N. Sivaranjani, MD Asst. Prof DR.N.SIVARANJA NI

2. DNA -the chemical basis of heredity - carries the genetic information found in chromosomes, mitochondria and chloroplasts  DNA is organized into genes - fundamental units of genetic information. Knowledge of the structure and function of nucleic acids is essential in understanding genetics and the genetic basis of disease. DR.N.SIVARANJANI

3. Central Dogma of Life DNA RNA Protein DR.N.SIVARANJANI

4. • DNA is a polymer of deoxyribonucleotides • Composed of monomeric units namely • Deoxyadenylate (dAMP) • Deoxyguanylate (dGMP) • Deoxycytidylate (dCMP) • Deoxythymidylate (dTMP) • The monomeric units held together by 3’,5’-Phosphodiester (PDE) bonds as back bone. DR.N.SIVARANJANI

5. Formation of phosphodiester bond DR.N.SIVARANJANI

6. Erwin Chargaff is biochemist (1905 - 2002) quantitatively analyzed DNA from different species. He found some crucial rule present in the DNA. He got Nobel prize for this at 1950 – 1954. Chargaff’s rule Edwin Chargaff DR.N.SIVARANJANI

7. Purine = Pyrimidines Single stranded DNA & RNAs do not obey rule Double stranded DNA & RNA (in some viruses) satisfies chargaff’s rule. DR.N.SIVARANJANI DR.N.SIVARANJA NI

8. James Watson Francis Crick DR.N.SIVARANJANI

9.  James Watson and Francis Crick – (1953)  Proposed - DNA as double helical structure  The informational content of DNA resides in the sequence in which the deoxyribonucleotides are ordered / arranged.  Salient Features 1.DNA is a right handed double helix – 2 polynucleotide chain twisted around each other on common axis Watson-Crick Model of DNA Structure DR.N.SIVARANJANI

10. 2. Two strands are Antiparallel - one strand runs in 5'-3' direction, other in 3'-5' direction 3. Width (or diameter) of a double helix is 20 Å (2 nm) 4. Each turn of helix is 34 Å (3.4nm) with 10 pairs of nucleotides, each pair placed at a distance of about 3.4 Å 5. DNA helix - Deoxyribose-PO4 – backbone – Hydrophilic, Nitrogenous bases – inside – Hydrophobic 6. Two polynucleotide chains are not identical but complementary to each other due to base pairing. DR.N.SIVARANJA NI

11. 7. The two strands are held together by Hydrogen bonds - A = T , G = C The hydrogen bonds are formed between a purine & pyrimidine 8. Major groove – wide PDE backbone Minor groove – Narrow PDE backbone 9. Complementary bp – Chargaff’s rule Adenine = Thymine Guanine = Cytosine 10. Genetic information resides on one of the two strands - Template strand or antisense strand or non coding opposite strand -Sense strand / Non template / coding strand Proteins interact with the exposed bases DR.N.SIVARANJANI

12. DR.N.SIVARANJANI

13. sugar-phosphate chain bases THE DOUBLE HELIX Width 20A° Each turn 34A° Each base pair 3.4A° 5’ 3’ 3’5’ Major groove Minor groove DR.N.SIVARANJANI

15. Traditionally, a DNA sequence is drawn from 5’ to 3’ end. A shorthand notation for this sequence is ACGTA DR.N.SIVARANJANI

16. The primary structure of DNA is the sequence 5’ end 3’ end 5’ 3’ Phosphodiester linkage DR.N.SIVARANJANI

17. Secondary structure of DNA is the double helix ( spiral stair case) DR.N.SIVARANJANI

18. • DNA exists in 6 forms - A,B,C,D,E and Z form. • B-form is most predominant form under physiological conditions. • A-form – Right handed helix , 11 bp per turn, tilting of bp by 20Å away from the central axis. • Z-form – Left handed helix, 12 bp per turn, move in ZIG-ZAG Conformations of DNA double helix DR.N.SIVARANJANI

19. A-DNA Characteristics property A-DNA Shape Broadest Type of helix Right handed Base pairs/turn 11 Rise / base pair 2.3 A° Helix diameter 25.5 A° Pitch/turn of helix 25.3 A° Major groove Narrow Minor groove Very broadDR.N.SIVARANJANI

20. B-DNA Characteristics property B-DNA Shape Intermediate Type of helix Right handed Base pairs/turn 10 Rise / base pair 3.4 A° Helix diameter 23.7 A° Pitch/turn of helix 34 A° Major groove Wide Minor groove Narrow DR.N.SIVARANJANI

21. Characteristics property Z-DNA Shape narrowest Type of helix Left handed Base pairs/turn 12 Rise / base pair 3.8 A° Helix diameter 18.4 A° Pitch/turn of helix 45.6 A° Major groove Flat Minor groove Very Narrow Z-DNA

22. Unusual Structures of DNA •Bent DNA • Adenine base containing DNA tracts – produce bend • Six adenosines in a row produce a bend of about 18⁰. • Important in the binding of some proteins to DNA. • Certain antitumor drugs (eg-cisplastin) produce bent structure in DNA. DR.N.SIVARANJANI

23. Triple standard of DNA due to additional hydrogen bonds between the bases Thymine forms two Hoogsteen hydrogen bonds to the adenine of A-T pair to form T-A-T. Cytosine forms two hydrogen bonds with guanine of G-C pairs that results in C-G-C. Triple helical structure is less stable than double helix - increased electrostatic repulsion.

24. Four-stranded DNA • High content of Guanine – form tetrameric structure called G-quartets. • These structures are planar & are connected by Hoogsteen hydrogen bonds. • Antiparallel four stranded DNA structures - G-tetraplexes. • Eukaryotic chromosomes - Telomeres are rich in guanine - forms G-tetraplexes. DR.N.SIVARANJANI

25. TYPES OF DNA DR.N.SIVARANJANI

26. Denaturation of DNA • ds DNA are held together by hydrogen bonds • Disruption of hydrogen bonds (by change in pH or increase in temperature) results in separation of strands • The phenomenon of loss of helical structure of DNA is known as denaturation • Phosphodiester bonds are not broken by denaturation. • It is measured by absorbance at 260nm. • ss DNA has a higher relative absorbance than ds DNA (Hyperchromatic effect)

27. Melting Temperature (Tm) • It is defined as the temperature at which half of the helical structure of DNA is lost. • G-C base pairs are more stable than A-T bp. • Tm is greater for DNAs with high content of GC. • Formamide destabilizes hydrogen bonds of base pairs - used in rDNA technology. Renaturation (reannealing): • It is a process in which the separated complementary DNA strands can form a double helix. • Renaturation is highly essential in the process of Replication.

29. Organization of DNA in cell • Prokaryotic DNA: • The DNA is organized as a single chromosome in the form of double stranded circle. • Packed in the form of nucleoids. • Eukaryotic DNA: • DNA is associated with various proteins - chromatin which then organized into compact structures - chromosomes. DR.N.SIVARANJANI

32. RNA • Single stranded Polymer of ribonucleotides held together by 3’5’ phosphodiester bonds. • Chemically less stable than DNA. • Presence of 2’-OH makes RNA more susceptible to hydrolytic attack (especially form Alkali) • Prone to degradation by Ribonucleases (Rnases)

33. • RNA base composition: • A + G ≠ U + C Chargaff’s rule does not apply (RNA usually prevails as single strand) • All types of RNA are generated by nuclear processing of a precursor molecule – Post transcriptional modification.

34. Major types of RNA Composition Functions Ribosomal RNA (rRNA) (very abundant) 50 - 80 % Integral part of ribosomes & act as a machinery for synthesis of proteins. Transfer RNA (tRNA) 10 - 20 % Carries activated amino acids to ribosomes. Messenger RNA (mRNA) 5 – 10 % Encodes sequences of amino acids in proteins.

35. mRNA • The template strand of DNA is transcribed into a single stranded mRNA by RNA polymerase enzyme. • It carries the message to be translated to a protein • Pre-m RNA or hnRNA on processing liberates functional mRNA which enter cytoplasm & take part in protein synthesis. • Shorter lifespan - quickly broken down after translation

36.  5’ Cap – • mRNA is capped by 7 methyl GTP at 5’ terminal end attached "backward" through a triphosphate linkage • stabilizes the mRNA, prevent the attack of 5’ exonuclease. • helps in recognition of mRNA for protein synthesis.  Coding region (introns) - which is translated to proteins • Initiating codon – AUG • Contains specific codon for different amino acids • Terminating codon – UGA , UAA, UAG. mRNA contains nucleotide sequence that is converted to a.a sequence of polypeptide chain in the process of translation.

37.  3’ Poly A tail : - Polymer of adenylate residues (20-250 nucleotides) – maintains intracellular stability by preventing attach of 3’ exonuclease. -Can be used to separate mRNA from other species of RNA. AUGUUUUACGCAUGCUAG

38. tRNA • They transfer amino acids from cytoplasm to the ribosomal protein synthesizing machinery • Soluble RNA molecule Varying in length from 74 – 95 nucleotides. • At least 20 species of tRNA in every cell corresponding to each 20 a.a required for protein synthesis. • Structure resembles clover leaf model- Robert Holley .

39. Unusual bases seen in tRNA –  Thymine,  Pseudouridine,  Dihydrouracil,  Hypoxanthine,  Methyl adenine,  Dimethyl Guanine. tRNA serves as an "adaptor" molecule that carries specific amino acid to the site of protein synthesis

40. • Acceptor arm – carriers amino acids has 7 base pair, capped with a sequence CCA (5’-3’) 3’ OH forms ester bond with COOH of a.a • DHU arm – dihydrouridine 3-4 base pair serve as recognition site for enzyme which adds a.a • Pseudouridine arm (TψC) – 5 base pair involve in binding of tRNA to ribosome

41. • Anti codon arm – 5 base pair recognizes the triplet nucleotide codon present in mRNA contains anticodon that base pair with codon of mRNA. (contains base sequences complementary to that of mRNA codon) responsible for the specificity of tRNA. For ex: mRNA contains AUG UUU UAC anticodon of tRNA UAC AAA AUG tRNA accepts the specific a.a coded by that codon of mRNA

42. Variable arm – tRNA divide into  class I – 75% , 3-5 bp  class II – 13-20 bp The nucleotides of codon has no affinity for a.a so tRNA act as adapters (mediates b/w mRNA & a.a)

44. rRNA • Nucleolus - rRNA is synthesized and assembled with proteins to form ribosome subunits. • Ribosomes provide necessary infrastructure for the mRNA, tRNA and amino acids to interact with each other for the translation. • Acts as a machinery for the synthesis of proteins.  4 different rRNA – 18 S, 5.8 S, 28 S & 5 S. • They are distributed in both 40S and 60S ribosomal subunits.

45. 80S Ribosomal RNA has catalytic activity. o Peptidyl transferase activity is carried out by 28S RNA which acts as a ribozyme. S = Svedberg units 2 8 50

46. DR.N.SIVARANJANI Types of RNA Functions Heterogeneous nuclear RNA (hnRNA) Serves as a precursor for mRNA Small nuclear RNA (snRNA) Involved in mRNA splicing Small nucleolar RNA (snoRNA) Involved in rRNA processing Small cytoplasmic RNA (scRNA) Involved in selection of proteins for export Transfer messenger RNA (tmRNA) Mostly present in bacteria. Promotes degradation of incorrectly synthesized proteins. Micro-RNAs (miRNAs) and Small Interfering RNAs (siRNAs) Inhibition of gene expression by decreasing specific protein production

47. Ribozymes Enzymes made up of RNA are called ribozymes Ribozymes or RNA enzymes are catalytic RNA molecules with sequence specific cleavage activity Ex: Spliceosomes contain ribozymes as well as protein components which serve to stabilize the structure of ribozymes. RNAse-P is another ribozyme, which generates the ends of tRNAs. Peptidyl transferase present in ribosomes - used for protein synthesis.

48. DNA RNA Site Nucleus Cytoplasm Strand Double Single Base pair Millions of bp 100-5000 bp Sugar Deoxy ribose Ribose Base A, G, C, Thymine A, G, C, Uracil Purine / pyrimidine content A = T , G = C . Obeys Chargaff’s rule. A ≠ U , G ≠ C Types A ,B ,C ,D, E & Z m RNA, t RNA, r RNA. Alkali hydrolysis Stable Susceptible Importance Carriers genetic information (Replication , Transcription) Protein synthesis (Translation)